Our previous work has demonstrated that the non-structural protein 1 (NS1) of influenza A virus is an accessory virulent factor implicated in the attenuation of the type I interferon (IFN) response of the host during viral infection. This is mainly achieved by the N-terminal dsRNA-binding region of the NS1 that prevents the induction of IRF-3, a key cellular transcription factor that when activated stimulates the IFN-beta promoter. Our recent results indicate that the NS1 of influenza B virus (less than 25% amino acid identity with the NS1 of influenza A virus) also prevents the induction of IRF-3 but in this case this is achieved by the concerted action of the N-terminal dsRNA-binding and the C-terminal non-dsRNA-binding regions. We are now planning to investigate at the molecular level the mechanism of action by which the NS1 proteins of influenza A and B viruses inhibit the activation of IRF-3 and antagonize the IFN response. Our preliminary data strongly suggest that sequestration of viral dsRNA by the NS1, a potent activator of IFN synthesis, is not entirely responsible for the IFN antagonism of these proteins, and identify two cellular RNA helicases as potential cellular targets for the influenza A virus NS1. Both of these RNA helicases, RIG-I and MDA-5, have recently been implicated in the induction of IFN through its ability to "sense" dsRNA and transmit an activation signal that results in IRF-3 phosphorylation. We will now investigate the role that these cellular RNA helicases play on the IFN antagonist function of the NS1 of influenza A virus and on modulation of virulence. In addition, we have designed experiments to understand the similarities and differences between the NS1 proteins of influenza A and B viruses with respect to their ability to inhibit the IFN response and to promote viral replication in the host. Our proposed experiments will generate novel information that not only will increase our knowledge on how influenza viruses disarm the antiviral IFN response, but that will also contribute to a better understanding on the role that cellular RNA helicases play in the initiation of the IFN response. Since the NS1 proteins are one of the determinants of virulence of influenza viruses, our proposal falls under one of the High-Priority Influenza Research Areas of the NIAID Biodefense Research Program: Determination of molecular basis of virulence of influenza viruses.